Preparation, physicochemical and biological evaluation of quercetin based chitosan-gelatin film for food packaging

Carbohydrate Polymers 227 (2020) 115348

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Preparation, physicochemical and biological evaluation of quercetin based chitosan-gelatin film for food packaging

T

Srasti Yadav, G.K. Mehrotra , Prabha Bhartiya, Anu Singh, P.K. Dutta ⁎

⁎

Polymer Research Laboratory, Department of Chemistry, Motilal Nehru National Institute of Technology Allahabad, Prayagraj, 211004, India

ARTICLE INFO

ABSTRACT

Keywords: Chitosan-gelatin films Quercetin Antibacterial Antioxidant UV protective

Ecofriendly chitosan-gelatin (Ch-ge) bio-composite films containing Quercetin-starch (Q) were synthesized using solution casting method. Physicochemical characteristics and mechanical properties of the resulting chitosangelatin containing Quercetin-starch films (Ch-ge-Q) were studied using UV, FTIR, XRD and SEM techniques. The films were also investigated for their swelling, water-vapor permeability (WVP), water solubility properties. The FTIR spectra confirmed the chemical interactions between the chitosan-gelatin and Q. Surface morphology of prepared film was analyzed by the SEM imaging while XRD spectra suggest the expanded crystallinity of the film with the addition of Q. The film also showed enhanced barrier property against UV rays. The reduction of watervapor permeability and increase in tensile strength while a decrease in elongation at break has been observed in the Ch-ge-Q film compared to Ch-ge film. The antibacterial activity of Ch-ge-Q film against both gram positive (B. substilis) and gram negative (E. coli) bacteria suggested the Q loaded Ch-ge films as more feasible antibacterial candidate especially against the strain E. coli. The antioxidant activity of the Ch-ge-Q film was evaluated using the DPPH and ABTS as standards and corresponded to 81.45% of DPPH and 72.2% of ABTS scavenging activities. It was observed that the film containing Quercetin-starch presented superior antioxidant activity results in comparison to Ch-ge film promising its application in food packaging.

1. Introduction With the accelerating stress to replace environmentally unsustainable petroleum based packaging materials, whilst still exhibiting promising shelf-life extension and food safety via spoilage due to pathogenic microorganisms, efforts have been intensified in recent decades towards developing active biodegradable biocomposite films based on natural protein and polysaccharide matrices (Lin, Abdel-Shafi, & Cui, 2019; Fortunati et al., 2018; Shankar & Rhim, 2018; Mujtaba et al., 2019; Wróblewska-Krepsztul et al., 2018). In the realm of active food packaging film materials, the literature is largely dominated by Chitosan, (Mitelut, Tanase, Popa, & Popa, 2015; Otoni, Espitia, AvenaBustillos, & McHugh, 2016; Gomez-Estaca, López-de-Dicastillo, Hernández-Muñoz, Catalá, & Gavara, 2014; Tripathi, Mehrotra, & Dutta, 2009; Hu, Jia, Zhi, Jin, & Miao, 2019) an abundantly available natural polysaccharide biopolymer. Chitosan has been also well recognized with some advantageous characteristics, including biocompatibility, hydrophilicity, biodegradability, non-toxicity, and bioadherence as well as nonantigenicity and cell affinity (Kumar, Dutta, & Sen, 2010; Singh et al., 2009). Chitosan and its derivatives have emerged essentially as an important constituent in the production of ⁎

both edible and non-edible types of functional food packaging films owing to its well-known non-toxic and anti-microbial biofunctions (Kumar, Kumari, Dutta, & Koh, 2013; Kumar, Vishwa, Kumari, & Dutta, 2016; Nigam, Kumar, Dutta, Pei, & Ghosh, 2016) coupled with exceptional film forming mechanical properties (Aguirre-Loredo, RodríguezHernández, Morales-Sánchez, Gómez-Aldapa, & Velazquez, 2016; Leceta, Guerrero, & de la Caba, 2013). Lately, focus has shifted to mindfully engineered advanced biocomposite materials in which both matrix and reinforcement are from bio-based origins. Another natural biomaterial gelatin, a protein obtained from partial hydrolysis of collagen found in skin and bones of marine and land animals, has been found to be an effective blending material in bio-composite film formation with polysaccharides such as cellulose, starch and chitosan owing to their similar biological characteristics (Sobral, Menegalli, Hubinger, & Roques, 2001; Suderman, Isa, & Sarbon, 2018). Apart from preventing lipid oxidation and dehydration of food materials, gelatin has been found to reinforce mechanical robustness and enhance the barrier properties of the resulting biocomposite films against light and atmospheric oxygen (Al-Hassan & Norziah, 2012; Etxabide, Coma, Guerrero, Gardrat, & de la Caba, 2017; Jahit, Nazmi, Isa, & Sarbon, 2016; Jamroz et al., 2018; Dammak, Bittante, Lourenco, & Sobral,

Corresponding authors. E-mail addresses: [email protected] (G.K. Mehrotra), [email protected] (P.K. Dutta).

https://doi.org/10.1016/j.carbpol.2019.115348 Received 9 August 2019; Received in revised form 7 September 2019; Accepted 18 September 2019 Available online 25 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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2017; Córdoba & Sobral, 2017; Kanmani & Rhim, 2014). Earlier, though the preparation and characterization of collagen-chitosan and gelatin –chitosan composite films have been reported, evaluation of their antimicrobial efficacy has not been investigated (Haghighi et al., 2019; Zhang, Liu, Ren, & Wang, 1997). Some works related to polymer nanocomposites such as ZnO/carboxymethyl cellulose nanocomposites (Upadhyaya et al., 2014), Fe3O4/alginate nanocomposites (Upadhyaya et al., 2015), ZnO grafted O-carboxymethylchitosan/curcumin-nanocomposite (Srivastava et al., 2012), chitosan/gelatin/Ag@ZnO bionanocomposites (Murali et al., 2019), Chitosan/PVA/ZnO) nanocomposites film (Kumar, Krishnakumar, Sobral, & Koh, 2018) have been recently reported by various researchers. Moreover, it has been demonstrated that with incorporation of additional ingredients such as natural plant based essential oils or phenolic compounds get fairly enhanced the antimicrobial, antioxidant and physicochemical properties of the resultant chitosan based composites (Akyuz et al., 2018; Yin, Yao, Cheng, & Ma, 1999). Bioflavonoids are among the major constituents of these plant extracts and from a biological perspective, one of the most extensively studied types of natural compounds. They tend to exert their antioxidant behavior by scavenging the free radicals that form during the oxidative processes (Ribeiro-Santos, Andrade, Melo, & Sanches-Silva, 2017). The literature survey has revealed that among the dietary flavonols, Quercetin (3,3′,4′5,7-Pentahydroxyflavone), is bestowed with a wellrecognized antibacterial (both against bacterial suspensions and surface attached bacterial biofilms) as well as antioxidant properties. It has been comparatively little investigated as a component of active food packaging film ingredient till date. In a recent systematic study of the inhibition activity of a large number of natural and synthetic flavonoids towards Staphylococcus aureus, Quercetin has been found remarkably active against the chosen bacteria (Hirai et al., 2010; Manner, Skogman, Goeres, Vuorela, & Fallarero, 2013; Yanishlievaa, Marinovaa, & Pokorny, 2006). Despite proven biocidal properties only a handful of studies on incorporation of Quercetin into biopolymer based films are available till date (Arcan & Yemenicioğlu, 2011; Basu et al., 2017; Benbettaïeb, Karbowiak, Brachais, & Debeaufort, 2015; Dutta, Tripathi, & Dutta, 2012; Silva-Weiss et al., 2018). Further, incorporating Quercetin-starch complex (Q) is likely to facilitate the interaction between positively charged ammonium groups of chitosan and functional groups present in Q via hydrogen bonds and electrostatic attractions. It is noteworthy that despite noticeable current spurt in the research activities in the field of active biodegradable biocomposite films, fundamental issues such as composition, mechanical strength, moisture retention and gas sorption properties among others remain a challenge to be addressed adequately. In this issue, the multilayer component systems, e.g., some polymers as the outer layers and other biopolymeric layers as the inner layers have an excellent biocompatibility, antimicrobial and immobilised surface property showed better behaviours compared with individual components one has drawn the attention of researchers for various kinds of specific applications (Najafabadi, Keshvari, Ganji, Tahriri, & Ashuri, 2012). In above context, and in continuation of our work (Singh, Dutta, Kumar, Kureel, & Rai, 2018, 2018b) on the subject, we have chosen two naturally occurring biopolymers chitosan (Ch) and gelatin (ge) and incorporated Quercetin-starch (Q) into it to synthesize their hybrid films by employing simple solution casting technique. The resulting Chge-Q film has been investigated for its structural composition, surface morphology, UV protection property, water vapor permeability and mechanical properties like elongation at break and tensile strength. The antimicrobial and antioxidant properties of the as synthesized composite film have also been evaluated to ascertain the overall efficacy of the film as prospective greener substitute for petroleum based plastic food packaging materials.

2. Materials and method 2.1. Materials Chitosan (higher molecular weight > 350 kDa and 79% deacetylated) was taken from Central Institute of Fisheries Technology (CIFT, Cochin). Glacial acetic acid (CAS Number 64-19-7), methanol (CAS Number 67-56-1), ethanol (CAS Number 64-17-5) and Tween 80 (CAS Number 9005-65-6) were purchased from Merck, India. Starch (CAS Number 9005-25-8) and gelatin (CAS Number 9000-70-8) were purchased from CDH, India. Quercetin (CAS Number 6151-25-3) was purchased from SRL, India. Potassium per sulphate (CAS Number 772721-1) was obtained from CDH. ABTS (2, 2- azinobis-3-ethylbenzothiazoline-6-sulphonic acid) (CAS Number 30931-67-0) and DPPH (1, 1diphenyl-2-picrylhydrazyl) (CAS Number 1898-66-4) were procured from SRL, India. Nutrient agar (LOT Number 0000241777) and nutrient broth (LOT Number 3011012) both were purchased from Himedia, Mumbai, India. The bacterial strains gram negative E. coli (MTCC Number 433) and gram positive bacteria B. subtilis (Gram + ve, MTCC Number 121) were purchased from IMTECH, Chandigarh, India. Milli-Q was obtained from our laboratory and used as solvent during the research. 2.2. Methods 2.2.1. Preparation of chitosan-gelatin (Ch-ge) based films incorporated with Quercetin-starch based complex (Q) The modified chitosan-gelatin based films containing Quercetinstarch based complex (Q) was prepared by following the procedure as described elsewhere (Priyadarshi & Negi, 2017). Firstly, we have prepared Quercetin-starch based complex. For the preparation of Quercetin-starch based complex we have taken 0.16 g Quercetin dissolved in 6 mL of ethanol and 0.01 mol/L Tween 80, further 2 mL of 1% starch solution were added and then mixture were continuously stirred for 5 h. The pellets and the supernatant were collected by centrifugation at 9000 rpm. Afterword, the collected pellets were washed with absolute ethanol for 2 times and dried at 40 °C for 24 h. They were stored in sealed containers prior to use. For the preparation of Ch-ge-Q film the 2% chitosan solution was prepared. Firstly chitosan dissolved with 1% glacial acetic acid and solution was stirred continuously overnight. Furthermore, for the preparation of 1% gelatin solution we have taken 1 g of gelatin in 100 mL distilled water and then added into the dissolved chitosan solution. The above synthesized Quercetin-starch based complex (1 mg/mL) was added into the chitosan-gelatin solution and the solution was stirred overnight. This solution (30 mL) was casted on petriplates by solution casting method and then dried at 50 °C for 12 h in an oven. The films were removed from petriplates and stored in air tight containers at room temperature (30 ± 2 °C). 2.2.2. Physicochemical and biological evaluation of Ch-ge-Q films 2.2.2.1. Water solubility and degree of swelling of the films. For the calculation of swelling degree and water solubility of the films, the film samples (2 × 2 cm) were cut into pieces (Peng, Wu, & Li, 2013; Tripathi, Gupta, & Dutta, 2013). To get the initial dry mass (w1) the film sections were dried at 105 °C. Then, the films pieces were positioned in 100 mL beakers with filled 50 mL of distilled water and enclosed with plastic wrappers and stored at room temperature for 24 h. Further, to get the final dry mass (w2) the films were dried out with filter papers. Then, the water solubility of films was analyzed using the given equation:

Film solubility =

w1

w2 w1

× 100

(1)

For the determination of swelling test a pre weighed dry samples

2

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were immersed in 50 mL beakers with 30 mL of distilled water at 25 °C for a given interval. Weight was taken of highly swollen samples by using filter paper for removing extra water. Same experiment was performed for three times, and the average value was measured as swelling ratio. The degree of swelling of repeated the film was analyzed using the given equation:

Film swelling degree =

w2

w1 w1

× 100

spectrophotometer cells. 2.2.2.6. FTIR analysis. The FTIR spectra were used to analyze the structural interactions of chitosan-gelatin films containing Q. The FTIR spectra of chitosan based films were recorded in the frequency range from 4000 to 400 cm−1 at resolutions of 8 cm−1using KBr pellets on Nicolet 170 SXFT-IR spectrophotometer.

(2)

2.2.2.7. X-ray diffraction analysis. The crystallinity of prepared samples were determined by X-ray diffraction patterns performed in the range of 5°–80° and 4°min−1 scan rate on Rigaku Smart lab diffractometer.

In the above formula w1 and w2 are the weights of dry and wet film samples respectively. 2.2.2.2. Mechanical properties. The standard method ASTM D882, used for the measurement of tensile strength (TS) and elongation at break (EAB), evaluated with the help of electronic tensile testing machine (Tinius Olsen Testing Machine) (ASTM, 1995). Before measurement, for the equilibration, film samples were cut into strips (2.5 × 10 cm) and had been placed for three days at 25 °C and 50% relative humidity (RH). The Film strips were fixed with holds, initial grip separation and crosshead speed were set to 50 mm and 50 mm/min at 25 °C until rupture. A stress-strain curve was record using a computer. TS were expressed in terms of MPa and EAB in proportion (%). Measurements had been repeated at least 3 times for each kind of film.

2.2.2.8. Scanning Electron Microscopy (SEM) analysis. The microstructure of the surface of chitosan-gelatin film containing Q was analyzed by SEM Using Zeiss (IIT Kanpur, Kanpur). 2.2.2.9. DPPH radical-scavenging activity. The DPPH radical scavenging assay was used for the determination of antioxidant activity of chitosan based films according to the method of (Singh, Dutta et al., 2018, 2018b). chitosan based films had been dissolved in 1% acidic acid solution. We have taken 1.0 mL of 0.25 μM of solution of DPPH in methanol and added into the different concentrations of chitosan based films (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg/mL). The experiment was taken place at 25 °C in the dark medium for 60 min and then with the help of UV–vis spectroscopy the absorbance was measured at wavelength 517 nm. The same experiment conditions process was applied for the blank and standard solution. The antioxidant capacity of DPPH radical scavenging activity was express as a % of inhibition in DPPH was calculated based on the following equation:

2.2.2.3. Water vapor permeability. Gravimetrically methodology used for determination of the water vapor permeability (WVP) of films, employing a changed ASTM E00996-00 procedure with some modifications (ASTM, 2001). For every measurement, we have taken entirely dried silica gel and then film pattern was cut and wrapped over a glass cup. The cup filled with distilled water was placed in a desiccator. Under room temperature, weight changes of the cup (to the nearest 0.001 mg) have been recorded as a function of time every 4 h for three successive days. WVP was estimated by the using given equation：

W×x WVP = t×A× P

DPPH radical scavenging activity (%) =

(3)

OP =

OPTR × L P

× 100

(6)

2.2.2.10. ABTS.+ scavenging activity. The antioxidant activity of chitosan based films determine by the another method ABTS.+ assay according to the previously described method (Singh, Dutta et al., 2018, 2018b). In this study, we had prepared ABTS.+ by taking 7 mM of 10 mg of ABTS and 2.6 mL of potassium per sulphate solution (2.45 mM) and then mixing properly. The prepared solution was take placed in dark condition at room temperature for 12–16 h to generate free radicals (ABTS.+) of ABTS. And further to maintain an absorbance of 0.70 ± 0.02 at wavelength 734 nm. We had taken 1 mL of prepared solution was diluted with 50 mL of Milli-Q water. We had added 3 mL of ABTS.+ solution into different concentrations of chitosan based films (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg/mL). For incubation at room temperature the experiment was taking placed for 1 h. Finally, using a UV–vis spectrophotometer (UV-2450, Shimadzu, India) the absorbance was measured at 734 nm. For the base line, Milli-Q water was used and the scavenging activity of ABTS was also calculated by method of Eq. (6).

2.2.2.4. Oxygen permeability (OP). According to the Crank (1979) with some modifications, the oxygen permeability (OP) was measured. The Chitosan based films were cut into strips (2 × 2 cm) for the OP test. Taken bottles (10 × 50 mm), and the mouth of the bottles was covered and sealed with the strip. Consequently, the bottles were placed in the desiccator at room temperature. The weight of weighing bottles were monitored every 1 d over 3 d. The slope of each line was determined by linear regression (R2 > 0.99) of weight change vs. time. The OPTR (Oxygen Permeability Transmission Rate) and OP (Oxygen Permeability) were calculated by using given Eqs. (4) and (5)

Slop Film Area

A1 A0

Where A0 is the absorbance of the standard solution (ascorbic acid), and A1 is the absorbance of the chitosan based films.

Where, W = weight gain (g) of film samples and = permeation area (m2), x = thickness (m) of the film, t was the lapsed time (s) for the weight gain of film, and P = Difference in partial vapor pressure between the pure water and dry atmosphere (2339 Pa at 20 °C). The WVP results of the films were expressed in form of g m−1 s-1 Pa−1. The test was performed in triplicate for each film.

OPTR =

A0

2.2.2.11. Antibacterial activity. The Agar well diffusion method was used for the determination of antibacterial activity of chitosan based films against gram positive bacteria B. substilis and gram negative bacteria E. coli according to previously described study (Naz et al., 2018). We have taken agar and broth, both dissolved in distilled water separately. Agar (14 g) dissolved in 500 mL of distilled water and nutrient broth (1.3 g) in 100 mL of distilled water and then all prepared (nutrient broth, agar and petri plates) had been sterilized in an autoclave at 121 °C ( ± 1 °C), 15 psi for 15 min. Sterilized nutrient broth and agar had been placed in a laminar chamber. Afterwards, Nutrient broth was dispersed onto solidified agar plate and it was used as a growing medium for the bacteria Escherichia coli (E. coli) and

(4) (5)

P = Difference in partial vapor pressure between the pure water and dry atmosphere (0.02308 atm at 25 °C), L represents the average film thickness. Measurements were determined in triplicate for each sample. 2.2.2.5. UV–vis spectroscopy analysis. The UV–vis analysis was conducted with a Double beam spectrophotometer (Shimadzu UV2450), using slit width of 2 nm and equipped with quartz 3

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Fig. 1. Preparation of chitosan-gelatin based films containing Quercetin-starch based complex (Q).

Bacillus subtilis (B. subtilis). In agar plates, holes had been created by sterilized test tube, and then the prepared bacteria medium was deposited on to agar plate and in the end 100 μL of every sample solution was placed in the wells of agar plates which were created in the center of the agar plate. This incubation was completed for 12 h at 37 °C ( ± 1 °C) and lastly the antibacterial activity of the films was analyzed by measuring the zone of inhibition (ZOI).

gelatin and chitosan (a cationic polysaccharide) aqueous mixtures exhibit enhanced gelation properties compared to the neat components solutions (Wang, Virgilio, Wood-Adams, & Heuzey, 2018, 2018b). However the modified chitosan-gelatin (gelatin is the only non-carbohydrate (polysaccharide) thickening agent used in food) based films containing Quercetin-starch based complex (Q) is not reported earlier as per our knowledge. The structural features of Chitosan and gelatin as object of study, including Quercetin-starch (Q) and their characteristics properties are shown in 3 steps of Fig. 1 expected to satisfy the best production and biological evaluation of the films as potential food packaging.

3. Results and discussion There have been numerous reports in the literature of using modified chitosan or derivatives for food packaging applications. As such 4

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OeH stretching that overlaps in the same region with NeH stretching. The peak at 1548 cm−1 may be assigned to NeO asymmetric stretch while the small peak near at 1641 cm−1 signifies the C]O stretching (amide I). The peaks at 1405 cm−1 and 2863 cm−1 indicate to OeH bending and CeH stretching respectively (Puttipipatkhachorn, Nunthanid, Yamamoto, & Peck, 2001). The FTIR spectra of Q are shown in Fig. 3. The peak of Quercetin at 1605 cm−1 may be consigned to C]C stretching vibration of phenyl ring, in a same plane with CeO stretching vibration observed at 1310 cm−1 (Singh, Dutta et al., 2018, 2018b). The FTIR spectra of Ch-ge film containing Q displays slight shifting of some existing bands which is evidence the addition of Q. The small modification was occur in the spectrum of Ch-ge-Q film when the incorporation of Quercetin-starch i.e. small shifting of amino and carbonyl bands. This results of spectrum shows that the hydrogen bonding between Q, gelatin and chitosan molecules which is presented in the reported result (Yin et al., 1999). The spectra of Ch-ge-Q films generated some new bands with slight shifting at 3264 cm−1 represents NeH stretch and also the spectrum band at 2863 cm−1 was related to NeO stretch, whereas bands at 1548 cm-1 and 1152 cm−1 represents the presence of NeO stretch and CeO stretch respectively. Moreover, the addition Q into Ch-ge increased the intensity of Ch-ge-Q film. Above results showed that the incorporation of Q in the composite films was compatible with Ch-ge matrix.

Fig. 2. UV–vis spectra of (a) Ch-ge film and (b) Ch-ge-Q film.

3.1. UV–vis spectroscopy Barrier properties are the most critical factors to decide the efficacy of film for packaging applications. Among barrier properties UVshielding films has gained enormous attention in recent years, since UV light can adversely affects quality of foods by generation of free radicals. In this context, UV-filters are the mainstay for good UV-protective films. To evaluate the UV-protection of Quercetin-starch (Q) incorporated Ch-ge films, films were scanned in 200–700 nm regions. Both Ch-ge film and Q incorporated film shows high absorption in UV–vis region. Ch-ge film shows absorption in 200–400 nm while Q loaded film shows slightly enhanced absorption in 200–400 nm region as well as extended absorption in 400–500 nm region also (Fig. 2). The extended absorption in 400–500 nm region after Q loading suggest successful incorporation of Q in Ch-ge film (Gómez-Guillén, Ihl, Bifani, Silva, & Montero, 2007). High absorption in UV region foster filtration of high energy UV rays, hence less exposure of UV rays to packed material.

3.3. XRD To design a particular application in the field of packaging industries crystallinity is one of the important criterions of the food packaging films. X-ray diffraction measurements were carried out to analyze of the nature of each film sample (Fig. 4). The XRD of Ch-ge film shows the amorphous nature of the films and the broad peak at centered around 21° of 2θ and the results are in line with the literature (Chen, Wang, Mao, Liao, & Hsieh, 2008; Nagahama et al., 2009). The peaks at around 8°, 9°, 12°, 17°, 21°, 24°, 25° and 27° are clearly visible corresponding to the characteristics peaks of Q which also indicate the crystalline nature of Quercetin bounded starch (Jahit et al., 2016). In the XRD spectrum of Ch-ge-Q, the characteristic peak of Ch-ge at 21° has been shifted to a lower 2θ value of 18° which related to the (110) plane and at 8° which corresponds to (020) planes respectively which seems to be appeared as that of chitosan (Saud, Pokhrel, & Yadav, 2019). Ch-ge-Q film shows distinct peaks of Q at 8°, 11°, 18° and 22° with slight shift. These results revealed that there were good interaction and compatibility among different components in the produced

3.2. FTIR The FTIR spectra of synthesized Ch-ge and Ch-ge-Q films shown in Fig. 3. The FTIR spectrum shows a band at 3283 cm−1 attributing to

Fig. 3. FT-IR spectra for (a) Ch-ge film (b) Quercetin-starch (Q) (c) Ch-ge-Q film.

Fig. 4. XRD pattern of (a) Quercetin-starch (Q) (b) Ch-ge film and (c) Ch-ge-Q film. 5

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Fig. 5. SEM images of (a) Ch-ge film (b) Ch-ge-Q film.

resulting films. 3.4. SEM SEM (Scanning electron microscope) images of Ch-ge film and Chge-Q films are shown in Fig. 5 respectively. By the addition of Q the surface morphology of Ch-ge film was modified which shown in Fig. 5(b). In the SEM images, the surface of Ch-ge film without adding Quercetin-starch were comparatively even and smoother surface which displays arbitrarily distributed microstructure and also this structure of Ch-ge film showed that chitosan film particles could have not to be crystallize form and shows an amorphous nature of film. Although, addition of Q to the Ch-ge film the surfaces of film was changed and observed as irregular, uneven rough structure, which shown in Fig. 5 and also a similar work was reported on Piyada et. al (Piyada, Waranyou, & Thawien, 2013).

Fig. 6. Swelling property of Ch-ge and Ch-ge-Q film.

properties of the resulting chitosan based films were significantly affected. TS of the only chitosan film were 10.54 ± .0565 MPa. With the incorporation of gelatin and Q, the TS were increased to 16.10 ± .1414 and 17.11 ± .3464 MPa respectively. The reason of higher TS of chitosan based films might be due to better compatibility of chitosan and gelatin/Quercetin-starch. The change in structures of Ch-ge and Ch-ge-Q film might lead to the increase in the TS of films. Some structural changes of films containing gelatin/Q may be assumed caused that lead to the increase in the TS of the films (Leceta et al., 2013). Furthermore, the increase of TS could be associated with the more molecular interaction between the chitosan and Quercetinstarch. The distribution and density of intra and intermolecular interaction between the polymer backbones in the film matrix are mainly responsible for mechanical properties of films (Ojagh, Rezaei, Razavi, & Hosseini, 2010). Besides it, with the addition of gelatin/Q with Ch film, the EAB of the chitosan films reduced significantly. The EAB of pure chitosan film was 11.04 ± .0565%, which was decreased to 9.34 ± .4242 and 5.100 ± .3162% for Ch-ge film and Ch-ge-Q film respectively. It might be due to the interdiction of flexibility of chitosan based films, because of formation of the microspores and cavities in the films with the addition of Q which is clearly evident from the SEM image. The results of tensile strength after the films were kept in the presence of UV radiation: The results of the tensile strength of the films after the UV radiation, the tensile strength (TS) decreased and EAB (%) values increased in the respect of results before the UV radiation. TS of the only chitosan film were 3.754 ± .1069 MPa. TS of Ch-ge and Ch-ge-Q were obtained after UV radition were 6.444 ± .2960 and 8.345 ± .3134 MPa respectively. The EAB of pure chitosan film was 6.656 ± .3118%, which was

3.5. Water solubility Solubility of chitosan based films in water is an essential property and water resistant is mandatory condition for food packaging film. Incubated the films for 24 h in distilled water the Ch-ge film consequently changed its shape while the incorporation of Quercetin-starch (Q), the Ch-ge-Q film were found to retain their integrity. Addition of Q in Ch-ge film the water solubility decreased from 58.13% (Ch-ge film) to 41.23% (Ch-ge-Q film). The high water resistance property of Ch-geQ film was possibly due the interaction and miscibility of amino group present in gelatin and chitosan with phenolic compound of Q (Piyada et al., 2013). 3.6. Swelling properties The swelling ratio of Ch-ge film reduced with incorporation of Quercetin-starch (Q). The swelling rate versus time (h) study of the Chge film shows the maximum swelling rate of about 382%. Incorporation of Q resulted in lower swelling index due to hydrophobic nature of Q (220% at the end of 24 h) (Al-Hassan & Norziah, 2012). Due to the interaction between Q and chitosan molecules by hydrophobic or hydrogen bonding the swelling values decreased, which resulted in the reduction of water uptake by gelatin molecules (Fig. 6). Additionally, this also results in the less exposure of polar side-chain groups to water molecules. 3.7. Mechanical properties The stress-strain curve of chitosan based films is shown in Fig. 7. With the addition of gelatin and Q to the chitosan the mechanical 6

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Fig. 7. (A) Stress-strain curve of chitosan based films before UV radiation and (B) Stress-strain curve of chitosan based films after UV radiation.

6.739 × 10−6 and 3.582 × 10−6 cc/m·24 h·atm of Ch-ge and Ch-ge-Q films respectively. According to previous report (Mcdonnell, Greeley, Kit, & Keffer, 2016) the permeation of oxygen through chitosan films was related to the association of O2 with NH3+. The Quercetin-starch could transform the chitosan film structure, resulting in decreased the value of OP.

increased to 10.61 ± .3769 and 11.37 ± .07071% for Ch-ge film and Ch-ge-Q film respectively after UV radiation. Stress-strain curve of chitosan based films: 3.8. Water vapor permeability For food packaging film water vapor permeability (WVP) is an important parameter which is used to evaluate the capacity of the film to minimize the moisture transfer between food and surrounding environment of packaging film. The value of WVP affected by some important factors like structural and chemical properties of polymeric backbone, hydrophobic interaction in the film, and also concentration and type of the additives (Aguirre-Loredo et al., 2016).To maintain the freshness of foods, the value of WVP should be maintained as low as possible. The WVP of chitosan film was 10.1 × 10−8 g m−1 s−1 Pa−1. The behavior of WVP seen in Ch-ge film should be associated with the presence of hydroxyl and amino groups in chitosan backbones, which could provide binding sites for water molecules. Water vapor permeation across film takes place via two processesadsorption followed by diffusion steps. Due the presence of hydrophilic domains of Ch-ge film the water vapor was more easily adsorbed, the diffusion step was improved substantially. The comparison of Ch-ge film and Ch-ge-Q film showed that the lower water vapor permeability of Ch-ge-Q film ranging from 7.75 × 10−8 to 7.57 × 10−8 g m−1 s−1 Pa−1. The WVP results of films shown in Table 1.

3.10. Antioxidant activity of Ch-ge and Ch-ge-Q film In present study, DPPH assay and ABTS assay two methods used for the determination of antioxidant activity. 3.10.1. DPPH radical scavenging activity In our research, DPPH scavenging activity was used to analyze the activity of Ch-ge film and Ch-ge-Q film. The reducing ability of all samples was analyzed with the help of absorbance value at wavelength 517 nm. Samples concentration increases the resulted absorbance value decreased Fig. 8(A) shows the % scavenging activity of DPPH. At concentration 1 mg/mL the resulting radical scavenging activity of Ch-ge film and Ch-ge-Q film are 31.2% and 81.45% respectively. After adding Quercetin-starch (Q) in Ch-ge film the antioxidant activity of Ch-ge-Q film revealed that better antioxidant activity in comparison to that Chge film i.e. the antioxidant property of Ch-ge-Q film enhanced (Ghosh et al., 2015). 3.10.2. ABTS.+ scavenging activity For the determination of ABTS.+ scavenging activity, ABTS (2, 2azino-bis (3-ethyl benzothiazoline-6-sulfonate) is a blue green color compound which used in food industry to measure the antioxidant activity. Due to the presence of their antioxidant content it is decolorized. In this study the stabilization of unstable free radicals is faster in ABTS assay in comparison to that of DPPH assay. The resulting ABTS assay of Ch-ge film and Ch-ge-Q film at concentration 1 mg/mL were calculated to be 18.11% and 72.2% respectively which is shown in Fig. 8(B). After addition of Q in Ch-ge film the antioxidant activity of Ch-ge-Q film display better activity in comparison to that Ch-ge film i.e. the Antioxidant property of Ch-ge-Q film enhanced (Anand David, Arulmoli, & Parasuraman, 2016). The results of the antioxidant property of ABTS assay and DPPH assay shown in Supplementary Table 3.

3.9. Oxygen permeability (OP) Chitosan based films having a good barrier against the permeation of oxygen. According to the previous literature (Butler, Vergano, Testin, Bunn, and Wiles (1996))) it discussed that the Chitosan based films had similar OP values of the commercially available ethylene vinyl alcohol copolymer films or polyvinylidene chloride (PVDC), whereas OP values of polyethylene films were much higher. For that reason, Chitosan based films were appropriate to be used as packaging materials for pharmaceuticals and food which explained in previous paper (Park, Marsh, & Rhim, 2002). As shown in Table 1 the value of OP decreased after the addition of Quercetin-starch (Q), whereas gelatin showed negligible influences on value OP. The OP decreased from 7.0347 × 10−6 cc/m·24 h·atm of chitosan film and after the addition of gelatin and Quercetin-starch the ranges between

3.11. Evaluation of antibacterial activity The results of antibacterial activity of chitosan based films were evaluated against both bacterial strains (gram positive bacteria B. substilis and gram negative bacteria E. coli) are presented in Fig. S1. The concentration of film in 1% acetic acid was 0.5 mg/mL. It was observed that the 1% acetic acid showed no apparent activity and from the figure the (zone of inhibition) ZOI of Ch-ge-Q film was greater than other samples (Fig. S1). The results of antibacterial activity of Ch-ge and Chge-Q film showed that the better antibacterial activity of Ch-ge-Q film than Ch-ge film (Jaisinghani, 2017; Wang, Virgilio et al., 2018, 2018b). The zone of inhibition (mm) of the chitosan based film against B. subtilis

Table 1 WVP and Oxygen Permeability (OP) of the Chitosan based films: Samples

WVP X 10−8 (g m−1 s−1 Pa−1)

Oxygen Permeability (OP) X 10−6 (cc/m·24 h·atm)

Ch film Ch-ge film Ch-ge-Q film

10.12 ± 0.07071a 7.75 ± 0.01414b 7.57 ± 0.09192c

7.0347 ± 0.2401a 6.739 ± 0.4419b 3.582 ± 0.1393c

a,b,c

different letters in the same column indicate significant differences among formulations (p < 0.05). 7

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Fig. 8. (A) DPPH radical scavenging activity of Ch-ge film and Ch-ge-Q film (B) ABTS radical scavenging activity of Ch-ge film and Ch-ge-Q film.

Table 2 Table for Inhibition zone of Ch-ge film and Ch-ge-Q film. Test Cultures

1% acetic acid (mm)

Chitosan film (mm)

Ch-ge film (mm)

Ch-ge-Q film (mm)

Gram Positive B. subtilis Gram Negative E. coli

11 ± 2

17 ± 2

20 ± 2

24 ± 2

11 ± 2

19 ± 2

22 ± 2

25 ± 2

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and E. coli are presented in Table 2. 4. Conclusion In our research, biodegradable chitosan based active films incorporating gelatin and Quercetin-starch (Q) had been successfully developed and a simple eco-friendly solution casting method was used for preparation of films. The addition of Quercetin-starch (Q) into chitosan-gelatin (Ch-ge) had upgraded the surface morphology and water vapor barrier of biopolymer films. Quercetin-starch had enhanced the tensile strength of Ch-ge film. Furthermore, the antioxidant activity of the Ch-ge film was significantly enhanced with the addition of Quercetin-starch. Antimicrobial study of resulting films suggested that the potential of Quercetin-starch (Q) to act as an antimicrobial agent, and the zone of inhibition of the films increased against E. coli and B. subtilis. The obtained results from the produced films are crucial because it suggests that by the combining chitosan, gelatin and Quercetin-starch (Q), there is a possibility of developing antibacterial and antioxidant films which can have good physicochemical properties. These chitosan based films (Ch-ge-Q) could be useful to increase the shelf life of food products. Acknowledgements The authors gratefully acknowledge to the MNNIT Allahabad, for providing stipend to SY. For XRD, UV–vis spectroscopy the authors are thankful to CIR (Centre for Interdisciplinary Research), MNNIT Allahabad, Prayagraj. The authors are thankful to IIT Kanpur and MNIT Jaipur’s MRC (Material Research Centre) for SEM and FTIR analysis. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115348. References Anand David, A. V., Arulmoli, R., & Parasuraman, S. (2016). Overviews of biological importance of quercetin: A bioactive flavonoid. Pharmacognosy Reviews, 10, 84–89. Aguirre-Loredo, R. Y., Rodríguez-Hernández, A. I., Morales-Sánchez, E., Gómez-Aldapa, C. A., & Velazquez, G. (2016). Effect of equilibrium moisture content on barrier,

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Further reading

Dutta, P. K., Tripathi, S., Mehrotra, G. K., & Dutta, J. (2009). Perspectives for chitosan based antimicrobial films in food applications. Food Chemistry, 114(4), 1173–1182.

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